Accelerated photoreceptor differentiation of hiPSC-derived retinal organoids by contact co-culture with retinal pigment epithelium

Stem Cell Research 39 (2019) 101491

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Accelerated photoreceptor differentiation of hiPSC-derived retinal organoids by contact co-culture with retinal pigment epithelium

T

Tasneem Akhtara, Haohuan Xiea, Muhammad Imran Khana, Huan Zhaob, Jin Baoa, Mei Zhanga, , ⁎ Tian Xuea,c,d, ⁎

a

Eye Center, The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China b Department of Biological and Environmental Engineering, Hefei University, Hefei 230601, China c Chinese Academy of Sciences Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China d Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China

ARTICLE INFO

ABSTRACT

Keywords: Contact co-culture Photoreceptors Retinal pigment epithelium Retinal organoids

Retinal organoids (ROs) derived from human-induced pluripotent stem cells recapitulate the three-dimensional structure of retina, mimic human retinal development, and provide cell sources for pre-clinical retinal transplantation. Retinal pigment epithelium (RPE) is crucial for normal outer retinal physiology, including phagocytosis of shed photoreceptor outer segments and secretion of neurotrophic and vasculotrophic growth factors. However, whether ROs-RPE co-culture can improve the differentiation of photoreceptors in ROs in vitro remains unknown. Herein, primary mouse RPE cells were contact co-cultured with ROs at different time points. Our results revealed that the RPE cells accelerated photoreceptor differentiation in ROs, as the cross talk between the RPE and ROs promoted the stage specific expression of photoreceptor markers at different differentiation stages. Thus, we established an improved co-culture system based on modeling of human retina-RPE dynamics during retinogenesis for the evaluation of ocular therapies.

1. Introduction Neural retina and retinal pigment epithelium (RPE) are two adjacent structures in vertebrate eye, which interact with each other and play essential roles to maintain visual functions (Marmorstein, 2001). RPE is essential for the retinal development. Previous study revealed that an early ablation (embryonic day E10–11) of RPE in mouse retina resulted in disorganization of the retinal layer, disruption of eye growth and subsequent eye resorption. The ablation of RPE at a later stage (E11.5–12.5) allowed the eye to maintain retinogenesis, but the laminar structure of the retina was disrupted. The vitreous failed to accumulate and adults were anophthalmic or severely microphthalmic by the end of gestation period (Raymond and Jackson, 1995). Moreover, RPE plays vital roles for the protection and survival of photoreceptors (German et al., 2008). Important functions of RPE include the synthesis and maintenance of the inter-photoreceptor matrix, photoreceptor membrane turnover, transport of nutrients from the vascular choroid,

phagocytosis of membranous discs shed by photoreceptor outer segments (Bok, 1993). Degeneration or functional loss of RPE results in subsequent loss of photoreceptors, leading to vision loss or even blindness (Song et al., 2015). Therefore, neural retina-RPE interaction is critical not only during retinal development but also vital for visual function of adult retina. Retinal organoids (ROs) derived from human-induced pluripotent stem cells (hiPSCs), are three-dimensional (3D) retina-like structures which recapitulate the cell type and differentiation process of human fetal retina (Zhong et al., 2014; Oswald and Baranov, 2018). Advances in ROs differentiation provide new approaches to study and to treat the retinal degenerative diseases, including photoreceptor replacement and drug design (Zhong et al., 2014; Yin et al., 2016; Santos-Ferreira et al., 2016). However, ROs differentiation is highly inefficient, time consuming, and costly. Furthermore, the microenvironment of hiPSCs including cell-cell interaction, cell-matrix interaction, and biophysical and biochemical signals, can modify the growth and differentiation

Corresponding authors at: Eye Center, The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China. E-mail addresses: [email protected] (M. Zhang), [email protected] (T. Xue). ⁎

https://doi.org/10.1016/j.scr.2019.101491 Received 12 December 2018; Received in revised form 19 June 2019; Accepted 25 June 2019 Available online 02 July 2019 1873-5061/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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efficiency of ROs (Li and Xie, 2005; Morrison and Spradling, 2008). Thus, it is important to improve the in vitro differentiation process of ROs and to ensure the appropriate microenvironment to accelerate differentiation within shorter period of time. Given the role of the RPE in phagocytosis, visual cycle (Vancura et al., 2018), protection against photo-oxidation (Girotti and Kriska, 2004), and secretion of vital factors for anatomical integrity of the retina (Cao et al., 1999), we sought to co-culture ROs with the RPE in a direct contact co-culture model to investigate whether the RPE can improve and accelerate the differentiation of photoreceptors in ROs. After the successful sequential differentiation of hiPSCs into ROs, we contact co-cultured the resulting ROs with primary mouse RPE cell culture to determine the effect of RPE cells on the differentiation of progenitors derived from hiPSCs. Our data revealed that RPE cells are vital for accelerated differentiation of ROs into photoreceptors under the direct contact condition. Thus, we presented an accelerated and improved model of organoid differentiation for further investigation of the molecular mechanisms underlying ROs-RPE interaction, morphogenesis, pathogenesis, and to explore treatment strategies for ocular disease.

(RMM) consisting of 60% DMEM, 25% Ham's F-12, supplemented with 10% FBS (Thermo Fisher Scientific), 100 μM taurine (Sigma), 2% B27, 1 × NEAA, 1 × GlutaMAX supplement (Thermo Fisher Scientific), and 1% antibiotic-antimycotic. On day 90, RMM was switched to RMM2 containing 90% DMEM/F12-GlutaMAX supplemented with 10% FBS, 1% N2 supplement (Thermo Fisher Scientific), 1 × NEAA, 1% antibiotic-antimycotic, 100 μM taurine (Sigma). 2.4. Primary culture of mouse RPE Wild-type C57BL/6 pregnant mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Stock No. C57BL/ 6Cnc) and maintained in a 12 h light/dark cycle, with food and water provided ad libitum. Mouse pups (P15–20 old) were used for primary culturing of the RPE. Mice were anaesthetized with isoflurane (Veteast, RWD Life Science, China) and de-cervialized. Mouse RPE cells were harvested following the previously reported protocol (FernandezGodino et al., 2016) with some modifications. Briefly, whole eye was enucleated using curved scissors and simultaneously dipped in fresh basal medium (Dulbecco's Modified Eagle's Medium). Eyes were carried to the cell culture laboratory in fresh basal medium. The eyes were rinsed in 10× antibiotic solution in phosphate buffer solution (PBS) for 5 min at room temperature (RT). After rinsing with basal medium to remove extra antibiotics, the eye balls were dissected for RPE sheets. The RPE sheets of all eyes (~60) were centrifuged (Sorvall™ ST16 R, Thermo Scientific) at 500 ×g for 5 min to remove the basal medium. The resulting pellet was re-suspended in 3 mL of 0.25% (w/v) trypsin (Thermo Fisher Scientific) and incubated at 37 °C and 5% CO2 for 20–25 min to digest the RPE sheet. The suspension was aggressively pipetted to achieve uniform trypsinized RPE cell suspension, which was then diluted with DMEM supplemented with 20% (v/v) fetal bovine serum to neutralize the trypsin reaction. The cell suspension was passed through 60 μm cell strainer to obtain a uniform cell suspension, then centrifuged at 500 ×g for 5 min and cells pellet was re-suspended in RPE medium consisting of DMEM/F12, 1% N2 supplement, 1 × NEAA and 1% antibiotic-antimycotic. The cell suspension was seeded on 24well plates pre-coated with laminin L2020 (Sigma-Aldrich) over night at 37 °C and 5% CO2. We used at least 30 post-natal mouse pups (~60 eyes) to culture three wells of the 24-well plate to get > 80% (1.5 × 106 cells/cm2) confluence. On day 3 of the primary RPE culture, unattached cells and cellular debris were gently washed with PBS and provided with RPE medium.

2. Materials and methods 2.1. Ethics statement All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Science and Technology of China (USTCACUC1801027). Adult mice were housed three to five in a cage and maintained under a 12 h light/dark cycle with ad libitum access to food and water. Both male and female mice were used in this study. Mouse pups at birth were designated as postnatal day 0 (P0). 2.2. hiPSC culture The BC1-eGFP (Kusuma et al., 2013; Luo et al., 2018; Zou et al., 2012) hiPSC line was gifted by Dr. Linzhao Cheng, Johns Hopkins University, Baltimore, Maryland, USA, and H9 cell line was gifted byDr. Zibin Jin, Wenzhou Medical University, China, with the verification of normal karyotype and was contamination free. The cell line was cultured on Geltrex (Thermo Fisher Scientific) pre-coated culture dishes, provided with TeSR-E8 medium (Stem cell Technologies) and incubated at 37 °C, 5% CO2 in humidified incubator. Within 4–5 days cells reached 80% confluence and were treated with Accutase (Stem cell Technologies) to passage the cells. To improve the cell survival, 10 μM Y-27632 was added (Stem cell Technologies) to the TeSR-E8 medium on the first day of cell platting.

2.5. Co-culture method Within 5 days, the RPE culture reached almost 100% confluence and was then co-cultured with 5–7 ROs for another 2 weeks in the respective ROs medium. For contact co-culture, neuroretinal vesicles were separated manually from the 2D culture at W4 of differentiation and co-cultured with the confluent primary RPE cell culture at the same week for W6 (W4 + 2W co-culture) time point, co-cultured at W5 for W7 (W5 + 2W co-culture) time point and co-culture at W6 for W8 (W6 + 2W co-culture) time point. Only for W3, neuroretinal vesicles were separated manually from the 2D culture at W3 and co-cultured on the same week. Co-culture and control ROs were provided with RDM when co-cultured with RPE on early stage of differentiation (day 30 day 42), RMM when co-cultured during middle stage (day 42 – day 90) and with RMM2 when co-cultured on later stage of differentiation (day 90 to onward). No RPE medium was provided after co-culturing of ROs with RPE. After 2 weeks of co-culture, the RPE sheet adhered to the ROs was manually removed and ROs were analyzed by IHC, Western blotting, and qRT-PCR for the expression of early and late retinal markers. ROs > 20 weeks old were first cut into 4 or 5 pieces of about 500 μm size, 4–5 days prior to co-culture because when we co-cultured the whole ROs (age W20 or above) without cutting, they start to die in 2D co-culture due to diffusion-limited delivery of exogenous factors (e.g.,

2.3. Retinal organoid differentiation The hiPSCs line was differentiated into ROs, as reported previously (Zhong et al., 2014). In brief, hiPSCs were treated with dispase (Stem cell Technologies), and then cell scrapper was used to collect the small clumps and provided with mTeSR1 medium (Stem cell Technologies) and 10 μM Blebbistatin (Sigma) to produce small aggregates (day 0 of differentiation). On day 7 of differentiation, the small aggregates were seeded on Geltrex precoated dishes with a density of approximately 10 aggregates/cm2, and provided with neural induction medium (NIM). On day 16 of differentiation, NIM was switched to retinal differentiation medium (RDM) consisting 70% DMEM (Thermo Fisher Scientific) and 30% Ham's F-12 (Thermo Fisher Scientific), supplemented with 2% B27 supplement (Thermo Fisher Scientific), 1 × NEAA (Thermo Fisher Scientific), and 1% antibiotic-antimycotic (Thermo Fisher Scientific). On day 28 of differentiation, neural domains were separated manually with the help of a sharpened Tungsten needle under an inverted microscope and kept in suspension culture with RDM. On day 42 of differentiation, RDM was replaced with retinal maturation medium 2

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nutrients, oxygen) to the internal cells. After converting into smaller size ROs were able to sustain through whole time period of co-culture.

antibodies, for RCVRN mouse anti-rabbit (Cell signaling technology) and for RHO rabbit anti-mouse (Cell signaling technology) for 2 h at RT. Membranes were again washed with TBST (5 × 10 min) and visualized with enhanced chemiluminescence (ECL Plus Detection Kit; Invitrogen) and bands were detected using ChemiScope 5300 (Clinx Science Instruments, Shanghai, China).

2.6. Conditioned media Mouse primary RPE tissue were cultured in laminated wells of 12well plate with high cell density. RPE primary culture was provided with RDM and that conditioned media was collected after every 24 h. Conditioned media along with fresh RDM with 1:1 ratio (v/v) was provided to ROs (treatment group). In control group half medium was replaced with fresh RDM.

2.10. Quantification and statistical analysis For quantitative scoring, immunostaining images were processed using ImageJ software (US National Institutes of Health, https://imagej. nih.gov/ij/) and analysis of five areas (each 5800 μm2) per fluorescence image were performed by counting the number of positive cells. Western blot images of the control and co-cultured samples were captured with ChemiScope 5300 and quantified by ImageJ using the area measuring tool. After background subtraction, band intensity was averaged. Data pertaining to IHC, Western blotting, and qRT-PCR was statistically analyzed with GraphPad Prism v7.00 (GraphPad Software, San Diego California USA, www.graphpad.com/scientific-software/ prism/) using Student's unpaired t-tests for comparing two groups and ANOVA for comparing more than two groups followed by Tukey's multiple comparisons test. In all experiments, N indicates the number of individual experiments. A P-value of < 0.05 was taken as statistically significant. Data were plotted as means ± SEM, if not otherwise noted. Significant values were represented as: *P < .05, **P < .01 and ***P < .001, with ns = P > .05.

2.7. Immunohistochemistry The ROs were fixed in 4% paraformaldehyde (Sigma) in PBS at pH 7.2–7.4 for 25 min at RT, then washed with PBS (3 × 10 min), dehydrated with sucrose gradient (15% for 1 h at RT and 30% overnight at 4 °C), and finally incubated in optical cutting temperature (OCT) compound (Tissue-Tek®) overnight at 4 °C. The ROs were cut to 9 μm sections unless otherwise stated. RO sections were air dried for 1 h, and washed with PBS (3 × 10 min). Tissue sections were permeabilized and blocked in blocking buffer (1% BSA (Sangon) in PBS with 0.25% Triton X-100 (Sangon) and 10% serum (Gibco)) for 1 h at RT, then incubated with primary antibody (key resources table 1) diluted in the same blocking buffer at 4 °C overnight. The following day, the tissue sections on the glass slides were washed with PBS (3 × 10 min), followed by the addition of secondary antibody solution (key resources table 1) for 1 h at RT in the dark and again washed with PBS (3 × 10 min). Samples were incubated in DAPI (1:1000, Thermo Fisher Scientific) for 8 min, and then washed with PBS (3 × 10 min). Cover slips were mounted over the glass slides, then dried at RT and stored at 4 °C for microscopic observation. At each time point, identical conditions for immunostaining were used and fluorescence images were taken with an LSM 800 confocal microscope (Zeiss), unless otherwise stated.

2.11. Key resources Key Resources Table(s) for the study is provided in supplementary information for this paper. 3. Results 3.1. Generation of hiPSC-derived photoreceptors using 2D/3D differentiation

2.8. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)

To generate ROs efficiently, we used a 2D/3D differentiation process based on the same protocol used to differentiate hiPSCs into ROs (Fig. 1A) (Zhong et al., 2014). To investigate the differentiation process of ROs, we analyzed the gene expression profiles of differentiated ROs at week 8 (W8), W15, and W25 with photoreceptor progenitor markers CRX and RCVRN, rod photoreceptor markers NRL, GNAT1 and RHO, and cone photoreceptor marker OPN1LW/MW. Results demonstrated that the expression of CRX increased significantly at W15 and remained consistent at W25 (Fig. 1B). The expression of RCVRN transcript increased at W15 and W25 (Fig. 1C). The expressions of NRL, GNAT1, RHO and OPN1LW/MW progressively increased from W15 to W25 (Fig. 1D–G). The level of photoreceptor-associated markers and other retinal cell markers were further examined by immunostaining at different time points. In our ROs differentiation system, BRN3A (Fig. 1H), a marker for early born-retinal ganglion cells, was observed at W8. Photoreceptor progenitor marker RCVRN protein was initially expressed in the proximal part of the ROs (Fig. 1I), and then its level increased and segregated to its corresponding layer (Fig. 1J), mimicking the retinal lamination. The OTX2 was mainly observed in developing outer nuclear layer as well as in inner side of retinal epithelium at W17 (Fig. 1K). Late born-müller cells expressing RLBP1 were first observed at W17, and spread in the whole ROs at W25 (Fig. 1L and N), consistent with previous study (Zhong et al., 2014). Various other retinal markers, like GNAT1, RHO, OPN1LW/MW, bipolar cell marker CHX10 and horizontal marker PROX1 were also examined in ROs (Fig. 1M and N). This spatiotemporal pattern of ROs differentiation closely mimicked human retinal differentiation (Zhong et al., 2014). Therefore, our differentiation system provided an reliable in vitro retinal development model.

The ROs were harvested, and total mRNA was extracted using TRIzol® reagent (Thermo Fisher Scientific). Total mRNA (400 ng) was reverse transcribed with PrimeScript RT Master Mix (TaKaRa) into complimentary DNA (cDNA). The synthesized cDNA was then amplified with gene-specific primers (key resources table 2). Using a SYBR Green Master Mix (Roche) and ABI Prism 7000 apparatus, qRT-PCR was performed with actin as the loading control following the manufacturer's instructions. 2.9. Western blot analysis The ROs were harvested and washed twice with PBS. Samples were lysed with PBS Plus 1% Triton-X 100 lysis buffer supplemented with protease inhibitor cocktail (Biotool). Tissue lysates were subjected to strong pipetting, followed by sonication for 2 min at 30% intensity with 3 s on/off and incubation on ice for 20 min. The lysates were then centrifuged at 14,000 rpm for 10 min at 4 °C. The protein concentration of the supernatant was measured using BCA protein assay kit (GenStar) following the manufacturer's instructions. Protein loading buffer (5%, 250 mM Tris-HCl; pH 6.8, 10% SDS, 50% glycerol, 0.5% bromophenol blue, 5% β-mercaptoethanol) was added to the supernatant, which was then boiled at 100 °C for 5 min. Protein (20 μg) from the control and cocultured ROs were resolved by SDS-PAGE and transferred to a nitrocellulose blotting membrane (Pall). Membranes were blocked for 1 h in 5% fat-free milk containing Tris-buffered saline with 0.1% Tween 20 (TBST, pH 7.4) and incubated overnight with primary antibodies for RCVRN (Merck Millipore) and for RHO (Sigma-Aldrich). Membranes were washed with TBST (5 × 10 min) and incubated in secondary 3

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Fig. 1. Generation of hiPSC-derived photoreceptors using 2D/3D differentiation. A. Schematic depiction of the differentiation protocol using 2D/3D culture. Scale bar, 500 μm. B–G. qRT-PCR, gene expression analysis log2 fold change of CRX, RCVRN, NRL, GNAT1, RHO, and OPN1LW/MW in ROs at different time intervals i.e., W8, W15, and W25, (W15 and W25 normalized with W8), N = 3, each independent experiment contained ~3–6 ROs. H. IHC of BRN3A (red) and DAPI (blue) in ROs at W8. N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. I. IHC of RCVRN (red) and DAPI (blue) in ROs at W8. N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. J. IHC of RCVRN (red) and DAPI (blue) in ROs at W17. N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. K. IHC of OTX2 (red) and DAPI (blue) in ROs at W17. N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. L. IHC of RLBP1 (red) and DAPI (blue) in ROs at W17. N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. M. IHC of RCVRN (red), CHX10 (red) GNAT1 (red), and DAPI (blue) in ROs at W25. N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. N. IHC of RHO (red), OPN1LW/MW (red) RLBP1 (red), PROX1 (red), and DAPI (blue) in ROs at W25. N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. Results are means ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001, and ns = P > .05, one-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. ROs-RPE interaction enhanced early photoreceptor differentiation in ROs

MW at all these time points at this stage (data not shown). Taken together, RO-RPE interaction enhanced the differentiation process of the RO favoring an enriched population of early photoreceptor cells. To assess whether ROs-RPE co-culture can up regulate the level of other early-born retinal markers, markers like AP2α (amacrine cell maker), BRN3A (ganglion cell marker) and PROX1 (horizontal cell marker) were analyzed in the co-culture ROs. Immunoreactive signals of these markers at W5 (W3 + 2W co-culture) showed no-significant difference in control and co-culture group (Fig. 3A–D). The mRNA expression of these markers also showed the consistent results (Fig. 3E). The effect of RPE cells on ROs differentiation has also been analyzed at W6 (W4 + 2W co-culture), and found no difference in mRNA expression of these markers in either group (Fig. 3F). These data indicated that ROs-RPE co-culture specifically affected the photoreceptor differentiation in vitro, which may be due to the direct contact between photoreceptor and RPE physiologically.

The culture environment is important for the differentiation of ROs from hiPSCs (DiStefano et al., 2018). Lack of extrinsic factors and supporting microenvironment may hinder the development of cells during the early stages of differentiation (Bradford et al., 2005; Khanna et al., 2006). Thus, to provide a microenvironment close to the native retina, we co-cultured primary mouse RPE cells with multiple ROs. We successfully cultured primary mouse RPE cells as a monolayer sheet, which could reserve important properties such as intracellular microfilaments and intact tight junctions for proper function (Hu and Bok, 2014). To assess whether RPE cells maintained their morphology after 3 weeks of primary culture in our culture system, we immunostained the RPE cells with different RPE markers-OTX2, SOX9, MITF, and ZO1. The positive staining of RPE markers suggested that RPE cells sustained their morphological features and were appropriate to conduct co-culture studies (Fig. 2A). To investigate, whether the RPE cells play a key role in the specification and patterning of ROs into photoreceptors, we co-cultured ROs with RPE cells for 2 weeks at W3, W4, W5, and W6, and then analyzed the level of RCVRN and other retinal markers after two weeks (Fig. 2B and C). The co-cultured ROs at W5 (W3 + 2W co-culture) showed a higher RCVRN mRNA expression compared to control (W5), but no RCVRN immunoreactive signal was found in either group at this stage (data not shown). The immunohistochemistry (IHC) results of the cocultured ROs at W6 (W4 + 2W co-culture) showed a dramatic increase in the number of RCVRN positive cells as compared to the control (W6) (Fig. 2D). The number of RCVRN positive cells was increased at W7 (W5 + 2W co-culture) (Fig. 2E), whereas the effect of RPE cells on ROs decreased at W8 (W6 + 2W co-culture) and the RCVRN level became non-significant between control and co-culture groups (Fig. 2F). Our data demonstrated that the level of RCVRN in the control ROs were low at W5 and W6 but began to increase at W7 and were up-regulated at W8. In contrast, the level of RCVRN in co-cultured group was upregulated at W6 (W4 + 2W co-culture), the fold change difference between the control and co-culture groups was narrowed down at W7 (W5 + 2W co-culture), and turned to non-significant at W8 (W6 + 2W co-culture) (Fig. 2G). The differences of RCVRN mRNA expression between the control and co-cultured ROs at all these time points also showed the consistent trend (Fig. 2H). Consistently, western blot analysis of RCVRN level at all these time points also showed the similar results (Fig. 2I–K). Taken together, RPE cells increased the level of RCVRN considerably during the transient phase (between W5 and W7), as it has been reported that ROs initiate to express RCVRN at W7 during in vitro differentiation (Zhong et al., 2014). This indicated ROs-RPE interaction enhanced the early photoreceptor differentiation in ROs in vitro. Although the RPE enhanced the transcript expression of RCVRN, yet it had no effect on expression of mature photoreceptor markers (RHO and OPN1LW/MW) at this stage. Neither control nor co-culture ROs showed any immunoreactive positive signal of RHO and OPN1LW/

3.3. The effect of conditioned media on photoreceptor progenitors of ROs We next determined whether a direct interaction between the RPE and photoreceptors was essential to RPE-induced expression of retinal progenitors to generate functionally mature organoids. We therefore cultured ROs and RPE in separate culture dishes, and added primary RPE cell culture medium to the ROs culture every 24 h. In contrast, the conditioned media in the absence of direct contact between the RPE and ROs did not influence the expression of RCVRN at W6 (W4 + 2W ROs) and also at W7 (W5 + 2W ROs) (Fig. 3G). This may imply that the functional association between the RPE and ROs is dependent upon direct contact with each other. 3.4. Participation of RPE in accelerated differentiation of photoreceptors in later stages of ROs To analyze the effect of the RPE on differentiation of photoreceptor in later stages of ROs, as studies have indicated that RPE-derived factors are important for photoreceptor preservation (Zhong et al., 2014), we co-cultured ROs with RPE cells for 2 weeks at different time points (Fig. 4A), and then analyzed the expressions of different retinal and photoreceptor markers in ROs by IHC, qRT-PCR, and Western blot. The protein level and mRNA expression of RHO and OPN1LW/MW in cocultured ROs at W21 (W19 + 2W co-culture) showed no significant difference as compared to control (W21) (data not shown). Western blot analysis at W23 (W21 + 2W co-culture) showed a significant increase in the RHO protein level in co-cultured ROs lysates (Fig. 4B). The mRNA expression of RHO and OPN1LW/MW increased significantly in co-culture ROs at W23 (W21 + 2W co-culture), but RPE did not influence the mRNA expression of early photoreceptor progenitors and other late photoreceptor markers at this stage (Fig. 4C). Immunoreactivity assays in co-culture groups at W23 (W21 + 2W co-culture) and at W24 (W22 + 2W co-culture) revealed that RPE cells increased the number of immunoreactive positive cells of RHO and OPN1LW/MW as compared 5

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to control ROs (Fig. 4D, E, F and G). ROs co-cultured with RPE cells for 2 weeks at W25 (W23 + 2W co-culture) revealed no change in expression of RHO and OPN1LW/MW, where effect of RPE on ROs became non-significant (data not shown). Together, these results revealed that

the level of RHO and OPN1LW/MW in the control ROs were low at W22 and W23, but began to increase at W24 and were up-regulated at W25 where the effect of RPE on ROs became non-significant (Fig. 4H and J). In contrast, the effect of RPE on the level of RHO and OPN1LW/MW in

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Fig. 2. ROs-RPE interaction enhanced the early photoreceptor differentiation. A. Immunostaining of primary RPE cell culture OTX2 (green), SOX9 (green) MITF (green), ZO1 (green) (3 week old cell culture) N = 3 independent experiments. Scale bar, 20 μm. B. Schematic depiction showing the adherent culture (2D), suspension culture (3D), and primary mouse RPE cell culture, followed by the co-culturing of ROs. C. Schematic depiction of co-culture time points, co-culture period, and harvesting of ROs for analysis. D. Whole-mount immunostaining of RCVRN (red) and DAPI (blue) in control versus co-cultured organoids at W6 (W4 + 2 W co-culture). N = 3, each independent experiment contained 6 ROs. Scale bar, 20 μm. E. Whole-mount immunostaining of RCVRN (red) in control versus co-cultured organoids at W7 (W5 + 2 W co-culture), N = 3, each independent experiment contained 6 ROs. Scale bar, 20 μm. F. Whole-mount immunostaining of RCVRN (red) in control versus co-cultured organoids at W8 (W6 + 2 W co-culture), N = 3, each independent experiment contained 6 ROs. Scale bar, 20 μm. G. Average number of positive cells/5800 μm2, indicating the trend of RCVRN level in control versus co-cultured ROs at different time points. H. Log2 fold-change of mRNA expression, indicating trend of control and co-cultured ROs at different time points, all groups normalized with control (W5), N = 3, each independent experiment contained > 6 ROs. I. Western blot, the level of RCVRN protein in control versus co-cultured RO lysates at W6 (W4 + 2 W co-culture), and quantification of relative RCVRN protein level normalized to actin. N = 3, each independent experiment contained > 12 ROs. J. Western blot, the level of RCVRN protein in control versus co-cultured RO lysates at W7 (W5 + 2 W co-culture), and quantification of relative RCVRN protein level normalized to actin. N = 3, each independent experiment contained > 12 ROs. K. Western blot, the level of RCVRN protein in control versus co-cultured RO lysates at W8 (W6 + 2 W co-culture), and quantification of relative RCVRN protein level normalized to actin. N = 3, each independent experiment contained > 12 ROs. Results are means ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001, and ns = P > .05, unpaired Student's t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and for studying the fundamental principles involved during development and regeneration (Riera et al., 2016). Numerous studies have examined differentiation of hiPSCs or hESCs into retinal cells in vitro (Oswald and Baranov, 2018; Achberger, 2018); however, only rodent retina has been co-cultured with RPE cells (Kaempf et al., 2008). Here, we showed for the first time a co-culture platform to study the effect of RPE cells on the photoreceptor differentiation in ROs. Furthermore, our contact co-culture system may be helpful to study the effects of diseaserelated RPE cells on photoreceptors and visa-versa, as the interaction between the RPE and photoreceptors is reportedly bidirectional (Strauss, 2005). This system may also provide an efficient in vitro model to investigate photoreceptor development and pathophysiology. RPE cells secrete multiple pigment epithelium-derived factors and vascular endothelial growth factors, which are important for the normal function of photoreceptors (Jablonski et al., 2000). The growth and differentiation of 3D organoids from stem cells are modulated by the RPE induced microenvironment including biophysical and biochemical signals, cell matrices, and cell-cell interactions (Morrison and Spradling, 2008; Li and Xie, 2005). The anatomical, physiological and symbiotic relationship between RPE and the neural retina specifically photoreceptor precursors and maturing cells endorse the selection of RPE cells as good candidate for the induction of neural activity. RPE cells also promote and reorganize the 3D orientation of developing photoreceptors (Baharvand et al., 2006). Here, we determined that conditioned media derived from the RPE did not influence the expression of different photoreceptor markers, which indicated that direct contact between RPE and ROs was mandatory for this fruitful cross talk. This may be due to strong functional and inherited attachment of RPE and retina with each other (Lamb et al., 2007; Kusakabe et al., 2009). However, the mechanism of intercellular interaction mediating the material transfer and the intrinsic and extrinsic factors involved need to be further investigated. The selection of RPE cells in this study was a challenge as immortalized RPE cell lines lose certain critical characteristics important for its proper function (Shang et al., 2018). Obtaining human RPE cells is difficult due to limited donor resources (Hu and Bok, 2014). Here, we used primary mouse RPE cells which retained many important physiological features during in vitro co-culture (Hazim and Williams, 2018). RPE cells when seeded at low cell density lost their pigmentation, adapt fibroblast kind morphology and also lost the functional characteristics (Shang et al., 2018). We cultured RPE cells at higher cell density to obtain confluent RPE cell culture within 4 days because the morphology of RPE was crucial during the whole time period of contact co-culture in this study. Diffusion-limited transport of oxygen and

co-culture groups was up-regulated at W23 (W21 + 2W co-culture) and at W24 (W22 + 2W co-culture). The differences of RHO and OPN1LW/ MW mRNA expression between the control and co-cultured ROs at different time points also showed the consistent trend as shown in (Fig. 4I and K). Collectively, RPE cells promoted the level of RHO and OPN1LW/MW considerably during the transient phase (between W23 and W24) and the effect of RPE on ROs became non-significant at W25, where the level of RHO and OPN1LW/MW in control groups reached to its peak consistent with previous study (Zhong et al., 2014). These data suggested that ROs-RPE co-culture enhanced the level of RHO and OPN1LW/MW in later phase of ROs differentiation. The effect of ROs-RPE co-culture was also analyzed on H9 cells derived ROs at early and later stages of differentiation to verify whether the effect of RPE might be due to the variation of hiPSCs line or this effect was generalized among other pluripotent stem cell lines. The IHC results of the co-cultured ROs on H9 cell line derived ROs at W6 (W4 + 2W co-culture) showed a dramatic increase in the number of RCVRN positive cells as compared to the control (W6) (Fig. 4L). The RCVRN mRNA expression between the control and co-cultured ROs at this time point also showed the consistent results (Fig. 4M). The mRNA expression of RHO between the control and co-cultured ROs on H9 cells derived ROs at W20 (W18 + 2W co-culture) showed no significant difference and IHC revealed no immunoreactive positive signals of RHO in either group (data not shown). The IHC results on H9 cell derived ROs at W21 (W19 + 2W co-culture) showed a significant increase in the number of RHO positive cells as compared to the control (W21) (Fig. 4N). The mRNA expression of RHO between the control and cocultured ROs at this time point also showed the consistent results (Fig. 4O). The only difference between ROs derived from two different pluripotent stem cell lines was that H9 cell line derived ROs start to express RHO slight earlier (W21) than BC1-EGFP cell line derived ROs. Therefore, we concluded that the effects of ROs-RPE co-culture on photoreceptor differentiation may also be generalized among different pluripotent stem cell lines. 4. Discussion In the current study, we established a ROs-RPE contact co-culture system to improve photoreceptor differentiation in a stage-specific manner. Generation of ROs is crucial to study ocular diseases (Hung et al., 2017), retinogenesis (Völkner et al., 2016), drug screening (Hynds and Giangreco, 2013), and to serve as a source of human retinal cells and tissues for transplantation (Kruczek et al., 2017). ROs have great potential in stem cell-based therapy for retinal-related diseases 7

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Fig. 3. The effect of conditioned media on early photoreceptor differentiation in ROs. A. Whole-mount immunostaining of BRN3A (red) in control versus co-cultured organoids at W5 (W3 + 2W co-culture), N = 3, each independent experiment contained 6 ROs. Scale bar, 20 μm. B. Quantification from A, relative fold (RF) change of positive cells/5800 μm2 area (five areas per fluorescence image) expressing BRN3A in control versus cocultured ROs. C. Whole-mount immunostaining of PROX1 (red) in control versus co-cultured organoids at W5 (W3 + 2 W co-culture), N = 3, each independent experiment contained 6 ROs. Scale bar, 20 μm. D. Quantification from C, relative fold (RF) change of positive cells/5800 μm2 area (five areas per fluorescence image) expressing PROX1 in control versus cocultured ROs. E. qRT-PCR, gene expression analysis of AP2α, BRN3A and PROX1 at W5 (W3 + 2W co-culture) in control versus co-culture ROs N = 3, each independent experiment contained > 6 ROs. F. qRT-PCR, gene expression analysis of AP2α, BRN3A and PROX1 at W6 (W4 + 2W co-culture) in control versus co-culture ROs N = 3, each independent experiment contained > 6 ROs. G. Schematic depiction showing collection of conditioned medium from primary RPE cell culture and the transfer into ROs suspension culture every 24 h for 2 weeks. qRT-PCR, gene expression analysis of RCVRN at W6 (W4 + 2 W ROs) and at W7 (W5 + 2 W ROs) in control versus ROs which were provided conditioned media from primary RPE cell culture for 2 weeks, N = 3, each independent experiment contained > 6 ROs. Results are means ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001, and ns = P > .05, unpaired Student's t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

nutrients is another critical factor in in vitro co-culture models (Miranda et al., 2015). Cell proliferation during organoid growth further increases the demand for exogenous factors, which can lead to hypoxic stress and cell-cycle arrest (Hubbi and Semenza, 2015). The major challenge in creating a co-culture system in the current study was the big size of the ROs at the later stages of differentiation. To reduce the size of ROs, we split ROs of about 500 μm size and then co-cultured them with RPE cells under 2D culture conditions, where they remained healthy. The differentiation of hiPSCs into photoreceptors is a long, tedious, labor-consuming, and expensive process. In this study, we improved the

differentiation efficiency of hiPSCs into photoreceptors. The efficiency may be further improved potentially by combining with other methods. A lack of important features such as vascularization, smooth muscle activity, and immune cells (e.g., microglia) are common hurdles in achieving functionally mature 3D organoids (Yin et al., 2016; Dutta et al., 2017; Takebe et al., 2013). The vascularization of ROs may overcome the limited diffusion of nutrients and oxygen/CO2 as 2D coculture model system here further limited the supply of oxygen/CO2 and nutrients, and the co-culture was unable to functionally sustain in 2D culture for longer period of time. Vascularization of ROs may provide a better platform for ROs differentiation in co-culture system, drug 8

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screening, and other in vitro studies. Further investigation will be performed to figure out the effects of RPE on maturation of vascularized ROs.

5. Conclusions Our research showed efficient differentiation of hiPSCs into ROs using a 2D/3D culture system, which mimicked the in vivo retinogenesis of embryonic eyes. In this study, we developed an improved method for the generation of neural photoreceptors from hiPSC-derived

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Fig. 4. ROs-RPE interaction accelerated photoreceptor differentiation in later stage of ROs. A. Schematic depiction of co-culture time points, co-culture period, and harvesting of ROs for analysis. B. Western blot, the level of RHO protein in co-cultured versus control RO lysates at W23 (W21 + 2 W co-culture), normalized to actin, experiment contained > 8 ROs. C. qRT-PCR, expression analysis of different genes at W23 (W21 + 2 W co-culture) in control versus co-cultured ROs, N = 3, each independent experiment contained > 6 ROs. D. IHC of RHO (red) and DAPI (blue) in control versus co-cultured organoids at W23 (W21 + 2 W co-culture), N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. E. IHC of OPN1LW/MW (red) and DAPI (blue) in control versus co-cultured organoids at W23 (W21 + 2 W co-culture), N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. F. IHC of RHO (red) and DAPI (blue) in control versus co-cultured organoids at W24 (W22 + 2 W co-culture), N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. G. IHC of OPN1LW/MW (red) and DAPI (blue) in control versus co-cultured organoids at W24 (W22 + 2 W co-culture), N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. H. Average number of positive cells/5800 μm2, indicating the trend of RHO level in control versus co-cultured ROs at different time points. I. Log2 fold-change of mRNA expression of RHO, indicating trend of control and co-cultured ROs at different time points, all the groups normalized with control (W21). J. Average number of positive cells/5800 μm2, indicating the trend of OPN1LW/MW level in control versus co-cultured ROs at different time points. K. Log2 fold-change of mRNA expression of OPN1LW/MW, indicating trend of control and co-cultured ROs at different time points, all the groups normalized with control (W21). L. IHC of RCVRN (red) and DAPI (blue) of H9 cells-derived control versus co-cultured organoids at W6 (W4 + 2W co-culture), N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. M. qRT-PCR, gene expression analysis of RCVRN in H9 cells-derived control versus co-cultured organoids at W6 (W4 + 2W co-culture), N = 3, each independent experiment contained > 6 ROs. N. IHC of RHO (red) and DAPI (blue) of H9 cells-derived control versus co-cultured organoids at W21 (W19 + 2W co-culture), N = 3, each independent experiment contained 6 ROs. section size, 9 μm. Scale bar, 20 μm. O. qRT-PCR analysis of RHO expression in H9 cells-derived control versus co-cultured organoids at W21 (W19 + 2 W co-culture), N = 3, each independent experiment contained > 6 ROs. Results are means ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001, and ns = P > .05, unpaired Student's t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

UE018). The study was also supported by the Anhui Provincial Natural Science Foundation (1808085MH289, 1708085QC57).

retinal progenitors. We further demonstrated that RPE cells are vital for ROs differentiation as the ROs-RPE co-culture improved and accelerated the differentiation efficiency of ROs into photoreceptors under the direct contact model. This suggests that direct interaction between RPE cells and photoreceptors plays a key role in the physiologically-oriented differentiation of photoreceptors during retinogenesis in vitro. Our data demonstrated that neuroretina joined to RPE cells led to the improved development of photoreceptor markers during retinogenesis.

Contact for reagent and resource sharing Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Professor Tian Xue ([email protected]). Appendix A. Supplementary data

Acknowledgments

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.scr.2019.101491.

Thanks to Dr. Linzhao Cheng for the BC1-eGFP hiPSC line, and Dr. Zibin Jin for H9 cell line. T.A. was supported by Chinese Government Scholarship. Thanks to all members in X.T. lab for discussion.

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Declaration of Competing Interests The authors declare no competing or financial interests. Author contribution T.X. and M.Z. conceived the project, designed the experiments. T.A. performed the in vitro co-culture experiments and data analysis. H.X. differentiated hiPSC into ROs. M.I·K helped with the data analysis. H.Z and J.B gave input for data analysis. M.Z., T.A. and M.I·K wrote the manuscript with the input from all the authors. All authors edited and proof-read the manuscript. Funding The study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020603, XDPB10), National Natural Science Foundation of China (81790644, 31601134), and the National Key Basic Research Program of China (2016YFA0400900), User with Excellence Program of Hefei Science Center CAS (2019HSC10

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